In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light, constituting reflected or fluorescent light, emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam.
The positioning elements are usually equipped with sensors to ascertain the present mirror position. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen passes through the beam splitter and then arrives at the detectors.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture pinhole (called the “excitation pinhole’), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels by way of the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
The published German Patent Application DE 199 06 757 A1 discloses an optical arrangement in the beam path of a light source suitable for fluorescence excitation, preferably in the beam path of a confocal laser scanning microscope, having at least one spectrally selective element for coupling the excitation light of at least one light source into the microscope and for blocking the excitation light or excitation wavelength scattered and reflected at the specimen out of the light coming from the specimen via the detection beam path. For variable configuration with very simple design, the arrangement is characterized in that excitation light of differing wavelengths can be blocked out by the spectrally selective element.
Alternatively, an optical arrangement of this kind is characterized in that the spectrally selective element can be set to the excitation wavelength that is to be blocked out. Also stated in the aforesaid document is the fact that the spectrally selective element can be embodied as an acoustooptical tunable filter (AOTF) or an acoustooptical deflector (AOD).
The published German Patent Application DE 198 59 314 A1 discloses an arrangement of a light-diffracting element for the separation of excitation light and emitted light in a microscope beam path, preferably in a confocal microscope, and in particular in a laser scanning microscope, in which context both the excitation light and the emitted light pass through the light-diffracting element and at least one wavelength of the excitation light is influenced by diffraction, while other wavelengths emitted by the specimen pass through the element uninfluenced and are thereby spatially separated from the excitation light. The arrangement contains an AOTF.
The known scanning microscopes have the advantage of spectral flexibility as compared to scanning microscopes in which the separation of illuminating light and detected light is implemented with a beam splitter, since the acoustoopticai component can be set, by activation with sound waves of differing frequencies, to any desired optical wavelength for illuminating light or detected light. In addition, with these scanning microscopes the spectral separation is many times better than in scanning microscopes having beam splitters. The use of scanning microscopes having a beam splitter (which can be embodied, for example, as a neutral splitter) is preferred for reflective specimens, because of elevated light power losses in the acoustooptical components. Scanning microscopes having beam splitters are moreover considerably more economical.
Commercial scanning microscopes usually contain a microscope stand such as the one also used in conventional light microscopy. As a rule, confocal scanning microscopes in particular can also be used as conventional light microscopes. In conventional fluorescent incident-light microscopy, that portion of the light of a light source, for example an arc lamp, that comprises the desired wavelength region for fluorescent excitation is coupled into the microscope beam path by means of a color filter (called the “excitation filter”). Coupling into the beam path of the microscope is accomplished by means of a dichroic beam splitter that reflects the excitation light to the specimen while it allows the fluorescent light proceeding from the specimen to pass largely unimpeded. The excitation light scattered back from the specimen is held back with a blocking filter that is nevertheless transparent to the fluorescent radiation. Optimal combination of mutually coordinated filters and beam splitters into an easily interchangeable modular filter block has been usual for some time. The filter blocks are usually arranged in a revolving turret within the microscope, as part of so-called fluorescent incident-light illuminators, thus making possible rapid and easy interchange. A fluorescence device for inverted microscopes which contains a revolving mount for the reception of multiple fluorescence cubes which is mounted rotatably on a drawer is described e.g. in German Patent DE 44 04 186 C1.